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Each cell of our bodies and of all multicellular organisms contains several members of the tetraspanin family. Tetraspanins provide a network that facilitates membranal interactions, hence their involvement in multiple physiological interactions. Our studies focus on CD81, which is required for cell fusion and cell-cell interactions, functions that have been subverted by major human pathogens, including hepatitis C virus and Plasmodium. Current research is aimed at understanding the role of CD81 in metastasis.

Clinical Trials

Clinical and Pathologic Studies in Non-Hodgkin's Lymphoma and Hodgkin's DiseaseRecruiting

The purpose of this study is to characterize the molecular and cell biology of the tumor
cells in lymphoma.

Role of an arginine-lysine rich motif in maturation and trafficking of CD19.Biochemical and biophysical research communicationsVences-Catalán, F., Kuo, C., Levy, S.2015; 465 (3): 319-323

Abstract

Normal expression of CD19 on the surface of B cells requires the presence of the tetraspanin molecule CD81. Previous studies have shown that surface expression of CD19 is highly reduced in CD81-deficient mouse B cells and that it is completely absent in an antibody deficient human patient with a mutation in the CD81 gene. The current study explored the contribution of an arginine-lysine rich motif, present in the membrane-proximal cytoplasmic domain of CD19, for the maturation and trafficking of this molecule. We demonstrate that this motif plays a role in the maturation and recycling of CD19 but in a CD81-independent manner.

Abstract

Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD81(-/-) mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD81(-/-) spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-α and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8α(+) DCs. In conclusion, this study demonstrates that CD81-Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases.

Abstract

A homozygous mutation in a splice site of the CD81 gene was identified previously in a patient, as the cause in a case of common variable immune deficiency (CVID). CD19 expression is reduced in mice that lack CD81; however, B cells in this patient lacked completely CD19 surface expression. The mutation led to an absence of the CD81 protein on the cell surface and it was assumed that the CD81 protein was not produced. Here we demonstrate that a truncated human CD81 mutant (CD81mut) was actually produced, but retained intracellularly. We also demonstrate that the truncated CD81mut protein is in close proximity to the intracellularly sequestered CD19. However, this interaction does not enable normal CD19 maturation and surface expression. In addition, we show that specific domains of CD81 enable retrieval and trafficking of human CD19 to the cell surface. Finally, we demonstrate that surface expression of CD19 requires CD81, even in non-B cells.

Abstract

A case of a young girl diagnosed with an antibody deficiency syndrome serves to highlight the role of CD81 in B cell biology. Moreover, this case illustrates a fundamental function of the tetraspanin family, namely their association with partner proteins. Characterization of the patient's B cells revealed lack of surface CD19 although both of her CD19 alleles were normal. Further analysis determined that her antibody deficiency syndrome was due to a mutation in the CD81 gene, which did not enable expression of CD19 on the surface of the patient's B cells. Actually, the partnership of CD81 with CD19 and the dependency of CD19 for its trafficking to the cell surface expression were first documented in CD81-deficient mice. CD81 is a widely expressed protein, yet the mutation in the antibody-deficient patient impaired mostly her B cell function. CD81 is required for multiple normal physiological functions, which have been subverted by major human pathogens, such as hepatitis C virus. However, this review will focus on the function of CD81 in cells of the adaptive immune system. Specifically, it will highlight studies focusing on the different roles of CD81 in B and T cells and on its function in B-T cell interactions.

Abstract

Chronic HCV infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this comes from clinical observations of tumor regressions upon anti-viral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon anti-viral treatments. We expressed the lymphoma B-cell receptors (BCRs) as soluble IgGs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region (V) gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.

Abstract

Hepatitis C Virus (HCV) infection is a global public health problem affecting over 160 million individuals worldwide. Its symptoms include chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped RNA virus mainly targeting liver cells and for which the initiation of infection occurs through a complex multistep process involving a series of specific cellular entry factors. This process is likely mediated through the formation of a tightly orchestrated complex of HCV entry factors at the plasma membrane. Among HCV entry factors, the tetraspanin CD81 is one of the best characterized and it is undoubtedly a key player in the HCV lifecycle. In this review, we detail the current knowledge on the involvement of CD81 in the HCV lifecycle, as well as in the immune response to HCV infection.

Abstract

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2's interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421-645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50's ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.

Abstract

Follicular lymphoma is a monoclonal B-cell malignancy with each patient's tumor expressing a unique cell surface immunoglobulin (Ig), or B-cell receptor (BCR), that can potentially recognize antigens and/or transduce signals into the tumor cell. Here we evaluated the reactivity of tumor derived Igs for human tissue antigens. Self-reactivity was observed in 26% of tumor Igs (25 of 98). For one follicular lymphoma patient, the recognized self-antigen was identified as myoferlin. This patient's tumor cells bound recombinant myoferlin in proportion to their level of BCR expression, and the binding to myoferlin was preserved despite ongoing somatic hypermutation of Ig variable regions. Furthermore, BCR-mediated signaling was induced after culture of tumor cells with myoferlin. These results suggest that antigen stimulation may provide survival signals to tumor cells and that there is a selective pressure to preserve antigen recognition as the tumor evolves.

Abstract

Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.

Abstract

In B lymphocytes, the cell surface receptor CD38 is involved in apoptosis of immature B cells, proliferation and differentiation of mature B cells. Although CD38 has been establish as a receptor, its signaling has been only partially characterized. As a result of the lack of signaling motifs in the cytoplasmic domain, CD38 must use a co-receptor to induce signaling within the cell. Accordingly, CD38 has been associated with different receptors such as the T-cell receptor/CD3 complex on T cells, CD16 on natural killer cells and MHC class II molecules on monocytes. The CD19/CD81 complex has been proposed as a co-receptor for CD38 in human B lymphocytes, but little or no characterization has been performed in mice. In this study the contribution of the CD19/CD81 complex in murine CD38 signaling was evaluated. Proliferation assays were performed using CD19(-/-) or CD81(-/-) deficient mice; CFSE-labeled B lymphocytes from wild-type mice and CD19(-/-) , CD81(-/-) and CD38(-/-) deficient mice were stimulated with agonistic antibodies against CD38. Immunoprecipitation and immunofluorescence were also performed to detect protein-protein interactions. Our results indicate that the CD19/CD81 complex interacts with CD38 but this interaction is not required to induce proliferation in mouse B lymphocytes, suggesting that other receptors may contribute to the proliferation induced by CD38 in B lymphocytes.

Complementary costimulation of human T-cell subpopulations by cluster of differentiation 28 (CD28) and CD81PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICASagi, Y., Landrigan, A., Levy, R., Levy, S.2012; 109 (5): 1613-1618

Abstract

Cluster of differentiation 81 (CD81) is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells: Primary human naïve T cells are better costimulated by CD81, whereas the memory T-cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared with that by CD28. We found that IL-6 played a role in the activation of the naïve T-cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T-cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T-cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire.

Abstract

We designed a whole tumor cell vaccine by "loading" lymphoma tumor cells with CG-enriched oligodeoxynucleotide (CpG), a ligand for the Toll-like receptor 9 (TLR9). CpG-loaded tumor cells were phagocytosed, delivering both tumor antigen(s) and the immunostimulatory CpG molecule to antigen-presenting cells (APCs). These APCs then expressed increased levels of costimulatory molecules and induced T-cell immunity. TLR9 was required in the APCs but not in the CpG-loaded tumor cell. We demonstrate that T cells induced by this vaccine are effective in adoptive cellular therapy for lymphoma. T cells from vaccinated mice transferred into irradiated, syngeneic recipients protected against subsequent lymphoma challenge and, remarkably, led to regression of large and established tumors. This therapeutic effect could be transferred by CD4(+) but not by CD8(+) T cells. A CpG-loaded whole-cell vaccination is practical and has strong potential for translation to the clinical setting. It is currently being tested in a clinical trial of adoptive immunotherapy for mantle-cell lymphoma.

Abstract

The unique immunoglobulin idiotype expressed on the surface of B lymphoma cells can be used as an effective antigen in tumor-specific vaccines when fused to immunostimulatory proteins and cytokines. A DNA vaccine encoding for an idiotype antibody single chain Fv (scFv) fragment fused to the Tetanus Toxin Fragment C (TTFrC) has been shown to induce protective anti-tumor responses. Protein-based strategies may be more desirable since they provide greater control over dosage, duration of exposure, and in vivo distribution of the vaccine. However, production of fusion protein vaccines containing complex disulfide bonded idiotype antibodies and antibody-derived fragments is challenging. We use an Escherichia coli-based cell-free protein synthesis platform as well as high-level expression of E. coli inclusion bodies followed by refolding for the rapid generation of an antibody fragment - TTFrC fusion protein vaccine. Vaccine proteins produced using both methods were shown to elicit anti-tumor humoral responses as well as protect from tumor challenge in an established B cell lymphoma mouse model. The development of technologies for the rapid production of effective patient-specific tumor idiotype-based fusion protein vaccines provides opportunities for clinical application.

Abstract

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

Abstract

CD81 is a tetraspanin cell surface protein that regulates CD19 expression in B lymphocytes and enables hepatitis C virus infection of human cells. Immunohistologic analysis in normal hematopoietic tissue showed strong staining for CD81 in normal germinal center B cells, a cell type in which its increased expression has not been previously recognized. High-dimensional flow cytometry analysis of normal hematopoietic tissue confirmed that among B- and T-cell subsets, germinal center B cells showed the highest level of CD81 expression. In more than 800 neoplastic tissue samples, its expression was also found in most non-Hodgkin lymphomas. Staining for CD81 was rarely seen in multiple myeloma, Hodgkin lymphoma, or myeloid leukemia. In hierarchical cluster analysis of diffuse large B-cell lymphoma, staining for CD81 was most similar to other germinal center B cell-associated markers, particularly LMO2. By flow cytometry, CD81 was expressed in diffuse large B-cell lymphoma cells independent of the presence or absence of CD10, another germinal center B-cell marker. The detection of CD81 in routine biopsy samples and its differential expression in lymphoma subtypes, particularly diffuse large B-cell lymphoma, warrant further study to assess CD81 expression and its role in the risk stratification of patients with diffuse large B-cell lymphoma.

Abstract

Antibody fragments (scFvs) fused to luciferase reporter proteins have been used as highly sensitive optical imaging probes. Gaussia princeps luciferase (GLuc) is an attractive choice for a reporter protein because it is small and bright and does not require ATP to stimulate bioluminescence-producing reactions. Both GLuc and scFv proteins contain multiple disulfide bonds, and consequently the production of active and properly folded GLuc-scFv fusions is challenging. We therefore produced both proteins individually in active form, followed by covalent coupling to produce the intended conjugate. We used an Escherichia coli-based cell-free protein synthesis (CFPS) platform to produce GLuc and scFv proteins containing non-natural amino acids (nnAAs) for subsequent conjugation by azide-alkyne click chemistry. GLuc mutants with exposed alkyne reactive groups were produced by global replacement of methionine residues in CFPS. Antibody fragment scFvs contained a single exposed azide group using a scheme for site-specific incorporation of tyrosine analogs. Incorporation of tyrosine analogs at specific sites in proteins was performed using an engineered orthogonal tRNA-tRNA synthetase pair from an archaebacterium. The unique azide and alkyne side chains in GLuc and the antibody fragment scFv facilitated conjugation by click chemistry. GLuc-scFv conjugates were shown to differentiate between cells expressing a surface target of the scFv and cells that did not carry this marker.

Abstract

We investigated the ability of CpG-oligodeoxynucleotide to generate an anti-tumor CD8+ T-cell immune response and to synergize with passive antibody therapy. For these studies, we generated an antibody against the idiotype on the A20 B-cell lymphoma line. This antibody caused the regression of established tumors, but ultimately the tumors relapsed. The escaping surface IgG-negative tumor cells were resistant to both antibody-dependent cellular cytotoxicity and signaling-induced cell death. Addition of intratumoral CpG to antibody therapy cured large established tumors and prevented the occurrence of tumor escapees. The failure of the combination therapy in mice deficient for CD8+ T cells demonstrates the critical role of CD8+ T cells in tumor eradication. When mice were inoculated with 2 tumors and treated systemically with antibody followed by intratumoral CpG in just one tumor, both tumors regressed, indicating that a systemic immune response was generated. Although antibody therapy can eliminate tumor cells bearing the target antigen, it frequently selects for antigen loss variants. However, when a poly-specific T-cell response was generated against the tumor by intratumoral CpG, even large established tumors were cured. Such an immune response can prevent the emergence of antibody selected tumor escapees and provide long-lasting tumor protection.

Abstract

CD81 is a component of the CD19/CD21 co-receptor complex in B cells. However, the role of CD81 in B cell activation has not been clearly elucidated. Here, we demonstrate that Cd81(-/-) B cells stimulated via their B cell receptor fluxed higher intracellular-free calcium ion along with increased phosphorylation of spleen tyrosine kinase and phospholipase gamma 2. Additionally, Cd81(-/-) B cells responded to toll like receptor 4 stimulation with increased nuclear factor-kappa B activation, cell proliferation and antibody secretion compared with wild-type B cells. Cd81(-/-) mice also mounted a significantly higher immune response to T-independent antigens than their wild-type counterparts. Finally, analysis of Cd81(-/-) B cells that were generated by bone marrow transplantation into Rag1(-/-) mice confirmed that the hyperactive phenotype is not dependent on the CD81-deficient environment. Taken together, these results indicate that CD81 plays a negative role in B cell activation in vitro and in vivo.

Abstract

The pleiotropic receptor tyrosine kinase Kit can provide cytoskeletal signals that define cell shape, positioning, and migration, but the underlying mechanisms are less well understood. In this study, we provide evidence that Kit signals through Wiskott-Aldrich syndrome protein (WASP), the central hematopoietic actin nucleation-promoting factor and regulator of the cytoskeleton. Kit ligand (KL) stimulation resulted in transient tyrosine phosphorylation of WASP, as well as interacting proteins WASP-interacting protein and Arp2/3. KL-induced filopodia in bone marrow-derived mast cells (BMMCs) were significantly decreased in number and size in the absence of WASP. KL-dependent regulation of intracellular Ca(2+) levels was aberrant in WASP-deficient BMMCs. When BMMCs were derived from WASP-heterozygous female mice using KL as a growth factor, the cultures eventually developed from a mixture of WASP-positive and -negative populations into a homogenous WASP-positive culture derived from the WASP-positive progenitors. Thus, WASP expression conferred a selective advantage to the development of Kit-dependent hematopoiesis consistent with the selective advantage of WASP-positive hematopoietic cells observed in WAS-heterozygous female humans. Finally, KL-mediated gene expression in wild-type and WASP-deficient BMMCs was compared and revealed that approximately 30% of all Kit-induced changes were WASP dependent. The results indicate that Kit signaling through WASP is necessary for normal Kit-mediated filopodia formation, cell survival, and gene expression, and provide new insight into the mechanism in which WASP exerts a strong selective pressure in hematopoiesis.

Abstract

CD81 is a tetraspanin family member involved in diverse cellular interactions in the immune and nervous systems and in cell fusion events. However, the mechanism of action of CD81 and of other tetraspanins has not been defined. We reasoned that identifying signaling molecules downstream of CD81 would provide mechanistic clues. We engaged CD81 on the surface of B-lymphocytes and identified the induced tyrosine-phosphorylated proteins by mass spectrometry. This analysis showed that the most prominent tyrosine phosphorylated protein was ezrin, an actin-binding protein and a member of the ezrin-radixin-moesin family. We also found that CD81 engagement induces spleen tyrosine kinase (Syk) and that Syk was involved in tyrosine phosphorylation of ezrin. After engagement of CD81, it colocalized with ezrin and F-actin, and this association was disrupted when Syk activation was blocked. Taken together, these studies suggest a model in which CD81 interfaces between the plasma membrane and the cytoskeleton by activating Syk, mobilizing ezrin, and recruiting F-actin to facilitate cytoskeletal reorganization and cell signaling. This mechanism might explain the pleiotropic effects induced in response to stimulation of cells by anti-CD81 antibodies or by the hepatitis C virus, which uses this molecule as its key receptor.

Abstract

Two to three percent of the world's population is chronically infected with hepatitis C virus (HCV) and thus at risk of developing liver cancer. Although precise mechanisms regulating HCV entry into hepatic cells are still unknown, several cell surface proteins have been identified as entry factors for this virus. Among these molecules, the tetraspanin CD81 is essential for HCV entry. Here, we have identified a partner of CD81, EWI-2wint, which is expressed in several cell lines but not in hepatocytes. Ectopic expression of EWI-2wint in a hepatoma cell line susceptible to HCV infection blocked viral entry by inhibiting the interaction between the HCV envelope glycoproteins and CD81. This finding suggests that, in addition to the presence of specific entry factors in the hepatocytes, the lack of a specific inhibitor can contribute to the hepatotropism of HCV. This is the first example of a pathogen gaining entry into host cells that lack a specific inhibitory factor.

Abstract

The unique immunoglobulin (Ig) idiotype on the surface of each B-cell lymphoma represents an ideal tumor-specific antigen for use as a therapeutic vaccine. We have used an Escherichia coli-based, cell-free protein-expression system to produce a vaccine within hours of cloning the Ig genes from a B-cell tumor. We demonstrated that a fusion protein consisting of an idiotypic single chain Fv antibody fragment (scFv) linked to a cytokine (GM-CSF) or to an immunostimulatory peptide was an effective lymphoma vaccine. These vaccines elicited humoral immune responses against the native Ig protein displayed on the surface of a tumor and protected mice against tumor challenge with efficacy equal to that of the conventional Ig produced in a mammalian cell and chemically coupled to keyhole limpet hemocyanin. The cell-free E coli system offers a platform for rapidly generating individualized vaccines, thereby allowing much more efficient application in the clinic.

Abstract

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.

Abstract

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.

Abstract

The tetraspanin web is composed of a network of tetraspanins and their partner proteins that facilitate cellular interactions and fusion events by an unknown mechanism. Our aim was to unravel the web partnership between the tetraspanin CD81 and CD19, a cell surface signaling molecule in B lymphocytes. We found that CD81 plays multiple roles in the processing, intracellular trafficking, and membrane functions of CD19. Surprisingly, these different roles are embodied in distinct CD81 domains, which function in the different cellular compartments: the N-terminal tail of CD81 has an effect on the glycosylation of CD19; the first transmembrane domain of CD81 is sufficient to support the exit of CD19 from the endoplasmic reticulum, although the large extracellular loop (LEL) of CD81 associates physically with CD19 early during biosynthesis; and finally, the TM2 and TM3 domains of CD81 play a role in the transmission of signals initiated upon engagement of the LEL. The participation of distinct CD81 domains in varied functions may explain the pleiotropic effects of CD81 within the tetraspanin web.

Abstract

Tetraspanins are evolutionarily conserved membrane proteins that tend to associate laterally with one another and to cluster dynamically with numerous partner proteins in membrane microdomains. Consequently, members of this family are involved in the coordination of intracellular and intercellular processes, including signal transduction; cell proliferation, adhesion, and migration; cell fusion; and host-parasite interactions.

Abstract

We identified the human germinal center-associated lymphoma (HGAL) in gene-expression profiling studies of diffuse large B-cell lymphoma (DLBCL). The expression of HGAL correlated with survival in patients with DLBCL. The HGAL gene is the human homolog of M17, a mouse gene expressed specifically in normal germinal center (GC) B cells. We generated a monoclonal antibody against the HGAL protein and show that HGAL is expressed in the cytoplasm of GC lymphocytes and in lymphomas of GC derivation. Among 727 lymphomas tested by immunohistochemistry on tissue microarrays, HGAL staining was found in follicular lymphomas (103 of 107), Burkitt lymphomas (40 of 40), mediastinal large B lymphomas (7 of 8), and in DLBCLs (103 of 151). Most marginal zone lymphomas lacked HGAL staining. Lymphocyte-predominant Hodgkin lymphomas (12 of 17) and, surprisingly, classical Hodgkin lymphomas (78 of 107) were found to be positive. Hierarchical clustering of comparative immunohistologic results in DLBCLs demonstrates that the expression of HGAL is similar to 2 other GC-associated proteins, BCL6 and CD10, but different from 2 markers associated with a non-GC phenotype, MUM1/IRF4 and BCL2. The restricted expression and GC specificity of HGAL protein suggest that it may have an important role in the diagnosis of specific lymphomas, and, potentially in the identification of subtypes associated with different prognoses.

Abstract

We used BIAcore to analyze the kinetics of interactions between CD81 and hepatitis C virus (HCV) envelope proteins. We immobilized different forms of HCV envelope proteins (E1E2, E2, and E2(661)) on the sensor and monitored their interaction with injected fusion proteins of CD81 large extracellular loop (CD81LEL) and glutathione-S-transferase (CD81LEL-GST) or maltose binding protein (CD81LEL-MBP). The difference between the GST and MBP fusion proteins was their multimeric and monomeric forms, respectively. The association rate constants between CD81LEL-GST or CD81LEL-MBP and the E1E2, E2 or E2(661) HCV envelope proteins were similar. However, the dissociation rate constants of CD81LEL-MBP were higher than those of CD81LEL-GST. Interestingly, the dissociation rate constant of CD81LEL-GST from E1E2 was much lower than from E2 or E2(661). The interaction between both forms of the CD81LEL fusion proteins and the HCV envelope proteins best-fitted the "heterogeneous ligand" model. This model implies that two kinds of interactions occur between envelope proteins and CD81LEL: one is strong, the other is weak. It also implies that the heterogeneity is likely due to the HCV envelope proteins, which are known to form non-covalently linked heterodimers and disulfide-linked aggregate.

Abstract

The tetraspanin web represents a new concept of molecular interactions in the immune system. Whereas most surface immune-modulating molecules involve receptor-ligand interactions, tetraspanins associate with partner proteins and facilitate their lateral positioning in the membrane. Moreover, the same tetraspanin molecule can associate with different proteins depending on the cell type. Most importantly, members of this family tend to associate with each other, together with their partners, in membrane microdomains that provide a scaffold for the transmission of external stimuli to intracellular-signalling components.

Abstract

Our laboratories have focused on the role of the tetraspanin CD81 in the regulation of mitotic activity. Previously we have shown that antibodies directed against CD81 can block the proliferation of cultured retinal pigment epithelial (RPE) cells. The present study investigates the role of this protein by analyzing the structure of the adult retina in mice with a null mutation of cd81. Adult cd81(-/-) mice were produced by crossing two inbred strains, NIHS-BC/Tac and 129X1/SvJ, carrying the cd81 mutation as heterozygotes (+/-). Seven cd81(-/-) mice and 11 wildtype (cd81(+/+)) littermates were anesthetized and perfused with paraformaldehyde. The eyes were removed and processed for examination by light and electron microscopy. In general, the retinas of the cd81(-/-) mice appeared normal. However, upon close examination, there was an 18% increase in the number of RPE nuclei in the cd81(-/-) mice. The photoreceptor layer of the cd81(-/-) mice was significantly thinner than that of the wild-type mice, even though there was no difference in the total thickness of the retinas in the two groups of mice. At the electron microscopic level we did not observe any differences in cell-cell junctions in the retinas of the cd81(-/-) mice as compared to their wild-type littermates. These data support a role for CD81 controlling cell-cycle and the number of RPE nuclei in the mouse retina.

Abstract

Tetraspanins have been hypothesized to facilitate the organization of functional multimolecular membrane complexes. In B cells the tetraspanin CD81 is a component of the CD19/CD21 complex. When coligated to the B cell Ag receptor (BCR), the CD19/CD21 complex significantly enhances BCR signaling in part by prolonging the association of the BCR with signaling-active lipid rafts. In this study CD81 is shown to associate with lipid rafts upon coligation of the BCR and the CD19/CD21 complex. Using B cells from CD81-deficient mice we demonstrate that in the absence of CD81, coligated BCR and CD19/CD21 complexes fail to partition into lipid rafts and enhance BCR signaling from rafts. Furthermore, a chimeric CD19 protein that associates only weakly if at all with CD81 fails to promote the association of coligated BCR with lipid rafts. The requirement for CD81 to promote lipid raft association may define a novel mechanism by which tetraspanins function as molecular facilitators of signaling receptors.

Abstract

CD81 is a widely expressed tetraspanin that associates in B cells with CD19 in the CD19-CD21-CD81 signaling complex. CD81 is necessary for normal CD19 expression; cd81(-/-) B cells express lower levels of CD19, especially cd81(-/-) small pre-BII cells, which are almost devoid of surface CD19. The dependence of CD19 expression on CD81 is specific to this particular tetraspanin since cd9(-/-) B cells express normal levels of CD19. Furthermore, expression of human CD81 in mouse cd81(-/-) B cells restored surface CD19 to normal levels. Quantitative analysis of CD19 mRNA demonstrated normal levels, even in cd81(-/-) pre-BII cells. Analysis of CD19 at the protein level identified two CD19 glycoforms in both wild-type and cd81(-/-) B cells. The higher M(r) glycoform is significantly reduced in cd81(-/-) B cells and is endoglycosidase H (endo-H) resistant. In contrast, the low M(r) glycoform is comparably expressed in cd81(-/-) and in wild-type B cells and is endo-H sensitive. Because endo-H sensitivity is tightly correlated with endoplasmic reticulum localization, we suggest that the dependency of CD19 expression on CD81 occurs in a postendoplasmic reticulum compartment where CD81 is necessary for normal trafficking or for surface membrane stability of CD19.

Abstract

Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV is also the major cause of mixed cryoglobulinemia, a B-lymphocyte proliferative disorder. Direct experimentation with native viral proteins is not feasible. Truncated versions of recombinant E2 envelope proteins, used as surrogates for viral particles, were shown to bind specifically to human CD81. However, truncated E2 may not fully mimic the surface of HCV virions because the virus encodes two envelope glycoproteins that associate with each other as E1E2 heterodimers. Here we show that E1E2 complexes efficiently bind to CD81 whereas truncated E2 is a weak binder, suggesting that truncated E2 is probably not the best tool with which to study cellular interactions. To gain better insight into virus-cell interactions, we developed a method by which to isolate E1E2 complexes that are properly folded. We demonstrate that purified E1E2 heterodimers bind to cells in a CD81-dependent manner. Furthermore, engagement of B cells by purified E1E2 heterodimers results in their aggregation and in protein tyrosine phosphorylation, a hallmark of B-cell activation. These studies provide a possible clue to the etiology of HCV-associated B-cell lymphoproliferative diseases. They also delineate a method by which to isolate biologically functional E1E2 complexes for the study of virus-host cell interaction in other cell types.

Abstract

Hepatitis C virus (HCV) is the major cause for non-A, non-B hepatitis. Most HCV-infected individuals do not clear the virus resulting in a chronic infection that may potentially lead to liver cirrhosis and hepatocellular carcinoma. In addition to hepatic manifestations, HCV infection is associated with B cell lymphoproliferative disorders, including mixed cryoglobulinemia, usually a benign condition, and overt B cell lymphoma. A direct role of HCV infection in the genesis of these B cell lymphoproliferative disorders has been suggested initially by epidemiological studies and is supported by recent studies, which analyzed the monoclonal B cells that proliferate in these disorders. How HCV induces B cell lymphoproliferative disorders is still unclear, it is probably not due to direct change of phenotype in B cells after viral infection, but may be due to an HCV-antigen driven process. Support for this hypothesis comes from the analysis of monoclonal B cells found in these disorders, which use a restricted repertoire of immunoglobulin variable region genes that are similar to those used by B cells that secrete anti-HCV antibodies. The fact that monoclonal IgM is resolved in HCV-infected patients who responded to anti-viral treatment supports the linkage between antigen persistence and B cell proliferation. Finally, the linkage between benign B cell proliferation and overt lymphoma is supported by the identification of a pre-malignant B cell clone that subsequently converted to an overt B cell lymphoma. The molecular basis for viral induced B cell proliferation is still unknown. One possibility is that HCV stimulates the proliferation of monoclonal B cells via their HCV-specific B cell receptor (BCR) on the cell surface. Binding of the HCVenvelope proteins to a cellular ligand, CD81, may also enhance this antigen-driven process. A recent report on regression of splenic marginal zone lymphoma after anti-viral treatment with interferon and ribavirin has significantly strengthened the cause-effect relationship between HCV infection and lymphoma. Further studies should determine whether BCRs expressed on HCV-associated lymphomas, particularly those that regress in response to anti-viral therapy, bind HCV antigens that stimulate their proliferation.

Abstract

The majority of hepatitis C virus (HCV)-infected individuals progress from acute to chronic disease, despite the presence of a strong humoral immune response to the envelope glycoproteins E1 and E2. When expressed in mammalian cells, E1 and E2 form both noncovalently linked E1E2 heterodimers, believed to be properly folded, and disulfide-linked, high-molecular-weight aggregates that are misfolded. Previously, we identified 10 human monoclonal antibodies (HMAbs) that bind E2 glycoproteins from different genotypes. Here we demonstrate that one of these HMAbs, CBH-2, is unique in its ability to distinguish between properly folded and misfolded envelope proteins. This HMAb recognizes HCV-E2 only when complexed with E1. The E1E2 complexes recognized by CBH-2 are noncovalently linked heterodimers and not misfolded disulfide-linked, high-molecular-weight aggregates. The E1E2 heterodimers seen by CBH-2 no longer associate with the endoplasmic reticulum chaperone calnexin and are likely to represent the prebudding form of the HCV virion.

Abstract

Plasmodium sporozoites are transmitted through the bite of infected mosquitoes and first invade the liver of the mammalian host, as an obligatory step of the life cycle of the malaria parasite. Within hepatocytes, Plasmodium sporozoites reside in a membrane-bound vacuole, where they differentiate into exoerythrocytic forms and merozoites that subsequently infect erythrocytes and cause the malaria disease. Plasmodium sporozoite targeting to the liver is mediated by the specific binding of major sporozoite surface proteins, the circumsporozoite protein and the thrombospondin-related anonymous protein, to glycosaminoglycans on the hepatocyte surface. Still, the molecular mechanisms underlying sporozoite entry and differentiation within hepatocytes are largely unknown. Here we show that the tetraspanin CD81, a putative receptor for hepatitis C virus, is required on hepatocytes for human Plasmodium falciparum and rodent Plasmodium yoelii sporozoite infectivity. P. yoelii sporozoites fail to infect CD81-deficient mouse hepatocytes, in vivo and in vitro, and antibodies against mouse and human CD81 inhibit in vitro the hepatic development of P. yoelii and P. falciparum, respectively. We further demonstrate that the requirement for CD81 is linked to sporozoite entry into hepatocytes by formation of a parasitophorous vacuole, which is essential for parasite differentiation into exoerythrocytic forms.

Abstract

We previously demonstrated that hepatitis C virus (HCV) binds to human CD81 through the E2 glycoprotein. Therefore, expression of the human CD81 molecule in transgenic mice was expected to provide a new tool to study HCV infection in vivo, as the chimpanzee is the only species currently available as a laboratory animal model that can be infected with HCV. We produced transgenic mice expressing the human CD81 protein in a wide variety of tissues. We confirmed binding of recombinant E2 glycoprotein to the liver tissue as well as to thymocytes and splenic lymphocytes in the transgenic mice. We inoculated chimpanzee plasma infected with HCV into these animals. None of these transgenic animals showed evidence of viral replication. Furthermore, human CD81 transgenic mice that lack expression of endogenous mouse CD81 were also resistant to HCV infection. We conclude that expression of human CD81 alone is insufficient to confer susceptibility to HCV infection in the mouse. The presence of additional possible factors for HCV infection is discussed.

Abstract

A key issue in the development of the central nervous system (CNS) is understanding the molecular mechanisms regulating cell number. The present study examines the role of CD81 (previously known as TAPA, the target of the antiproliferative antibody) in the control of brain size and glial cell number. CD81 is a member of the tetraspanin family of proteins. This group of small membrane proteins is associated with the regulation of cell migration and mitotic activity. Glial cells express CD81, and antibodies directed against this protein suppress the mitotic activity of cultured cells. In this study, we examine the effects of the CD81 -/- mutation on the CNS of mature mice. These mice have extremely large brains, as much as 30% larger than the brains of wild-type (+/+) littermates. The increase in brain weight is accompanied by an increase in the number astrocytes and microglia, whereas the number of neurons and oligodendrocytes in the CD81 -/- animals appears to be normal. When the CD81 -/- mutation is placed on different genetic backgrounds, there is a remarkable range in the penetrance of the null allele phenotype, demonstrating that the mutation can be affected by modifier loci. This work provides support for the role of CD81 in the regulation of astrocyte and microglial number, perhaps by regulating cell proliferation by a contact inhibition-dependent mechanism.

Abstract

Hepatitis C virus (HCV) infection is associated with extrahepatic B-cell lymphoproliferative disorders. To determine whether a viral antigen drives this B-cell expansion, the B-cell receptors were cloned from HCV-associated lymphomas and were expressed as soluble immunoglobulins. The rescued immunoglobulins were then tested for their ability to bind the HCV-E2 envelope glycoprotein, an antigen that was previously implicated in the pathogenesis of HCV-associated B-cell diseases. One of 2 lymphoma immunoglobulin test cases bound the E2 protein in a manner identical to a bona fide human anti-E2 antibody. Moreover, it bound E2 from multiple viral genotypes, suggesting reactivity with a conserved E2 epitope. These findings support the hypothesis that some HCV-associated lymphomas originate from B cells that were initially activated by the HCV-E2 protein and might explain the association between HCV infection and some B-cell lymphoproliferative disorders.

Abstract

Since the genomic sequence of HCV was determined, significant progress has been made towards understanding the functions of the HCV-encoded proteins, despite the lack of an efficient in-vitro replication system or convenient small-animal model. The identity of the receptor for HCV remains elusive, however. Low-density lipoprotein receptor, CD81, and GAGs may all act as receptors for HCV, either sequentially or by different viral quasispecies. Recent work using pseudotypic VSV bearing E1 or E2 chimeric molecules showed that entry of the E1 pseudotype can be inhibited by recombinant LDLr, whereas the E2 pseudotype is more sensitive to inhibition by recombinant CD81 or heparin. These results suggest that E1 and E2 may be responsible for interactions with different cellular molecules. It is also conceivable that additional, yet unidentified, cellular proteins are involved in viral binding and entry. Intriguingly, the reports of HCV-RNA associated with PBMC suggest that HCV infection may not be restricted to hepatocytes. Thus, separate reservoirs of virus may exist, and HCV may use different receptors to access these different cell types.

Abstract

Dendritic cells (DCs) are important for the initiation of immune responses to foreign antigens. Their antigen uptake and presentation capacities enable them to prime and activate T cells. Immature DCs capture antigens; however, they must be activated to mature before serving as efficient antigen-presenting cells. The antigen-presenting capacity of DCs can be diminished during viral infection and as a consequence of tumor formation. Chronic infection with hepatitis C virus (HCV) has been shown to affect the allostimulatory function of DCs. In this study, it is demonstrated that monocyte-derived DCs from patients with chronic HCV infection do not respond to maturation stimuli. Instead, they maintain their immature phenotype, reflected by the pattern of cell surface markers and by their continued capacity to uptake antigen. Moreover, their allostimulatory abilities are impaired compared with those of mature DCs derived from healthy donors. To investigate a possible correlation between viral clearance and this DC maturation defect, patients with resolved HCV infection after a course of antiviral therapy were studied. Results demonstrate that DCs from patients who cleared HCV behaved like DCs from healthy donors: in response to maturation stimuli, they decrease antigen uptake, up-regulate expression of appropriate surface markers, and are potent stimulators of allogeneic T cells. (Blood. 2001;97:3171-3176)

Abstract

We examined the role of IL-18 in preventing the development of and in reversing established allergen-induced airway inflammation and airway hyperreactivity (AHR), the cardinal features of asthma. IL-18, which potently induces IFN-gamma, was administered into the respiratory tract as cDNA in a replication-deficient adenovirus (Adv). Treatment of OVA-sensitized mice with the IL-18-expressing Adv reduced allergen-specific IL-4 production, airway eosinophilia, and mucus production, increased IFN-gamma production, and prevented the development of AHR. The effects of the IL-18 Adv treatment were dependent on the presence of IFN-gamma and IL-12. Moreover, administration of the IL-18 Adv to mice with established AHR greatly reduced AHR and IL-4 production and increased IFN-gamma production. These results demonstrate that IL-18, when administered by Adv into the respiratory tract, effectively reduces AHR and replaces an established Th2-biased immune response with a Th1-biased response.

Abstract

CD81 is expressed on human T cells at all stages of development. CD81 is physically associated with CD4 and CD8 and antibodies against CD81 generate signals which influence thymocyte adhesion and proliferation. Here we evaluate the function of CD81 on mature T cells. We employ a system in which B cells present superantigen to autologous T cells and find that anti-CD81 promotes T cell-B cell collaboration. Anti-CD81 induces T cell-B cell adhesion of peripheral blood lymphocytes which is partially mediated by LFA-1. CD81 engagement promotes LFA-1-dependent T cell activation, IL-2 production and proliferation. The antibody 5A6 was uniquely potent in exerting these effects compared to another antibody to CD81 or to antibodies that react with other tetraspanins expressed on T cells, anti-CD53 or anti-CD82. CD81-derived signals rapidly induce high-avidity LFA-1 as measured by cell binding to recombinant ICAM-3-coated fluorescent microspheres or by cell adhesion to ICAM-3-coated plastic. 5A6 activation of LFA-1 does not expose the high-affinity conformation epitope recognized by monoclonal antibody 24.

Abstract

Hepatitis C virus (HCV)-associated B cell lymphomas were previously shown to express a restricted repertoire of immunoglobulin V(H) and V(L) genes, V(H)1-69 and VkappaA27, respectively. Although this suggests a role for antigen selection in the pathogenesis of these lymphomas, the driving antigen involved in the clonal expansion has not been identified. B cell response to a viral antigen, the HCV envelope glycoprotein 2 (E2), was analyzed in an asymptomatic HCV-infected patient. Single B cells, immortalized as hybridomas and selected for binding E2, were analyzed for their V gene usage. Sequences of these V region genes demonstrated that each hybridoma expressed unique V(H) and V(L) genes. Remarkably, these anti-E2 hybridomas preferentially used the V(H)1-69 gene. Analysis of replacement to silent mutation ratios indicated that the genes underwent somatic mutation and antigenic selection. In a separate report, human anti-E2 antibodies were also shown to express the same V(H) gene. These data strengthen the hypothesis that the HCV-associated lymphomas are derived from clonally expanded B cells stimulated by HCV.

Abstract

Vaccination with naked DNA encoding a specific allergen has been shown previously to prevent, but not reverse, the development of allergen-induced airway hyperresponsiveness (AHR). To enhance the effectiveness of DNA vaccine therapies and make possible the treatment of established AHR, we developed a DNA vaccination plasmid containing OVA cDNA fused to IL-18 cDNA. Vaccination of naive mice either with this fusion DNA construct or with an OVA cDNA-containing plasmid protected the mice from the subsequent induction of AHR. Protection from AHR correlated with increased IFN-gamma production and reduced OVA-specific IgE production. The protection appeared to be mediated by IFN-gamma and CD8(+) cells because treatment of mice with neutralizing anti-IFN-gamma mAb or with depleting anti-CD8 mAb abolished the protective effect. Moreover, vaccination of mice with preexisting AHR with the OVA-IL-18 fusion DNA, but not with the OVA cDNA plasmid, reversed established AHR, reduced allergen-specific IL-4, and increased allergen-specific IFN-gamma production. Thus, combining IL-18 cDNA with OVA cDNA resulted in a vaccine construct that protected against the development of AHR, and that was unique among cDNA constructs in its capacity to reverse established AHR.

Abstract

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.

Abstract

We demonstrated previously that CD81(-/-) mice have an impaired Th2 response. To determine whether this impairment affected allergen-induced airway hyperreactivity (AHR), CD81(-/-) BALB/c mice and CD81(+/+) littermates were sensitized i.p. and challenged intranasally with OVA. Although wild type developed severe AHR, CD81(-/-) mice showed normal airway reactivity and reduced airway inflammation. Nevertheless, OVA-specific T cell proliferation was similar in both groups of mice. Analysis of cytokines secreted by the responding CD81(-/-) T cells, particularly those derived from peribronchial draining lymph nodes, revealed a dramatic reduction in IL-4, IL-5, and IL-13 synthesis. The decrease in cytokine production was not due to an intrinsic T cell deficiency because naive CD81(-/-) T cells responded to polyclonal Th1 and Th2 stimulation with normal proliferation and cytokine production. Moreover, there was an increase in T cells and a decrease in B cells in peribronchial lymph nodes and in spleens of immunized CD81(-/-) mice compared with wild-type animals. Interestingly, OVA-specific Ig levels, including IgE, were similar in CD81(-/-) and CD81(+/+) mice. Thus, CD81 plays a role in the development of AHR not by influencing Ag-specific IgE production but by regulating local cytokine production.

Abstract

Hepatitis C virus (HCV) glycoprotein E2 binds to human cells by interacting with the CD81 molecule, which has been proposed to be the viral receptor. A correlation between binding to CD81 and species permissiveness to HCV infection has also been reported. We have determined the sequence of CD81 from the tamarin, a primate species known to be refractory to HCV infection. Tamarin CD81 (t-CD81) differs from the human molecule at 5 amino acid positions (155, 163, 169, 180, and 196) within the large extracellular loop (LEL), where the binding site for E2 has been located. Soluble recombinant forms of human CD81 (h-CD81), t-CD81, and African green monkey CD81 (agm-CD81) LEL molecules were analyzed by enzyme-linked immunosorbent assay for binding to E2 glycoprotein. Both h-CD81 and t-CD81 molecules were able to bind E2. Competition experiments showed that the two receptors cross-compete and that the t-CD81 binds with stronger affinity than the human molecule. Recently, h-CD81 residue 186 has been characterized as the critical residue involved in the interaction with E2. Recombinant CD81 mutant proteins were expressed to test whether human and tamarin receptors interacted with E2 in a comparable manner. Mutation of residue 186 (F186L) dramatically reduced the binding capability of t-CD81, a result that has already been demonstrated for the human receptor, whereas the opposite mutation (L186F) in agm-CD81 resulted in a neat gain of binding activity. Finally, the in vitro data were confirmed by detection of E2 binding to cotton-top tamarin (Saguinus oedipus) cell line B95-8 expressing endogenous CD81. These results indicate that the binding of E2 to CD81 is not predictive of an infection-producing interaction between HCV and host cells.

Abstract

Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

Abstract

We describe the use of a soluble CD81-Fc fusion protein to screen for novel monoclonal antibody (MAb) reactive with the extracellular loops of murine CD81 (TAPA-1). Two such MAbs, Eat1 and Eat2 (for Extracellular Anti-TAPA1), were used to assess the expression and function of CD81 on murine lymphocytes. Although CD81 is expressed uniformly on all human lymphocytes, murine CD81 was found to be expressed at much higher levels on resting B cells than on resting T cells. This was particularly evident when staining with the new MAbs, Eat1 and Eat2. The molecule is also functionally active on B cells, as Eat1 and Eat2 induce homotypic adhesion of B lymphocytes. Stimulated B cells undergo early apoptotic events in the presence of Eat2, as shown by binding of Annexin V-fluorescein isothiocyanate (FITC). Polyclonal activation of murine T cells also induces higher level CD81 expression, and many immortalized murine T-cell lines express high levels of the protein. In contrast to human CD81, which is expressed equally on all thymocytes, murine CD81 is induced during thymic development, being expressed at high levels on CD4+CD8+ thymocytes, in contrast to other subsets of thymocytes. Finally, murine dendritic cells, splenic macrophages, and non-killer (NK) cells all express high levels of CD81. We conclude that CD81 is differentially expressed in the murine immune system, and is involved in regulating the adhesion and activation of murine B cells.

Abstract

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT. The E2661-HATMCT chimeric glycoprotein was able to bind a number of conformation-dependent monoclonal antibodies and a recombinant soluble form of CD81, suggesting that it was folded in a manner comparable to "native" E2. Furthermore, cell surface-expressed E2661-HATMCT demonstrated pH-dependent changes in antigen conformation, consistent with an acid-mediated fusion mechanism. However, E2661-HATMCT was unable to induce cell fusion of CD81-positive HEK cells after neutral- or low-pH treatment. We propose that a stretch of conserved, hydrophobic amino acids within the E1 glycoprotein, displaying similarities to flavivirus and paramyxovirus fusion peptides, may constitute the HCV fusion peptide. We demonstrate that influenza virus can incorporate E2661-HATMCT into particles and discuss experiments to address the relevance of the E2-CD81 interaction for HCV attachment and entry.

Abstract

A truncated soluble form of the hepatitis C virus E2 glycoprotein, E2661, binds specifically to the surface of cells expressing human CD81 (hCD81) but not other members of the tetraspanin family (CD9, CD63, and CD151). No differences were noted between the level of E2661 binding to hCD81 expressed on the surface of rat RBL or KM3 cells compared to Daudi and Molt-4 cells, suggesting that additional human-cell-specific factors are not required for the primary interaction of E2 with the cell surface. E2 did not interact with African green monkey (AGM) CD81 on the surface of COS cells, which differs from the hCD81 sequence at four residues within the second extracellular region (EC2) (amino acids [aa] 163, 186, 188, and 196), suggesting that one or more of these residues defines the site of interaction with E2. Various recombinant forms of CD81 EC2 show differences in the ability to bind E2, suggesting that CD81 conformation is important for E2 recognition. Regions of E2 involved in the CD81 interaction were analyzed, and our data suggest that the binding site is of a conformational nature involving aa 480 to 493 and 544 to 551 within the E2 glycoprotein. Finally, we demonstrate that ligation of CD81 by E2661 induced aggregation of lymphoid cells and inhibited B-cell proliferation, demonstrating that E2 interaction with CD81 can modulate cell function.

Abstract

Rapid production of protein-based tumor-specific vaccines for the treatment of malignancies is possible with the plant-based transient expression system described here. We created a modified tobamoviral vector that encodes the idiotype-specific single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted scFv protein in the extracellular compartment. This material reacts with an anti-idiotype antibody by Western blotting, ELISA, and affinity chromatography, suggesting that the plant-produced 38C13 scFv protein is properly folded in solution. Mice vaccinated with the affinity-purified 38C13 scFv generate >10 micrograms/ml anti-idiotype immunoglobulins. These mice were protected from challenge by a lethal dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13 IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for generating tumor-specific protein vaccines may provide a viable strategy for the treatment of non-Hodgkin's lymphoma.

Abstract

This study was designed to test whether cytotoxic T cell (CTL) responses to DNA vaccination are dependent upon MHC class II-restricted priming of CD4+ T cells. Because DNA vaccination may directly transfect dendritic cells, and dendritic cells may be capable of directly stimulating CD8+ T cell responses, such priming might be unnecessary. To test this hypothesis, C57BL/6 mice were immunized intramuscularly or intradermally with DNA encoding either whole OVA, a class I (Kb)-restricted peptide epitope of OVA (amino acids 257-264, SIINFEKL), or this class I-restricted epitope plus the adjacent class II (I-Ab)-restricted epitope of OVA (amino acids 265-280, TEWTSSNVMEERKIKV). Very low to negligible CTL responses were observed in mice vaccinated with the SIINFEKL construct, whereas mice vaccinated with the SIINFEKLTEWTSSNVMEERKIKV or with the complete OVA construct made equally robust CTL responses. These responses were sensitive to blocking by anti-CD8 mAb and were shown to be SIINFEKL-specific by using SIINFEKL peptide-pulsed EL-4 cells as targets. To ensure that the generation of these CTL responses was indeed dependent upon CD4+ T cell help, mice were depleted of either CD4+ or CD8+ cells before immunization. Depletion of CD4+ cells completely abrogated the CTL response to OVA DNA, as did depletion of CD8+ cells. Thus, we conclude that the CTL response to both intramuscular and intradermal DNA vaccination is highly dependent upon the generation of CD4+ T cell help via a class II MHC-dependent pathway. These results will be relevant for the construction of minimal-epitope vaccines for DNA immunization.

Abstract

Tetraspanins (or TM4SF) are expressed in a wide variety of species and regulate cell adhesion, migration, proliferation and differentiation. We have identified and sequenced six new members of the tetraspanin family, called Tspan-1-6, from human cDNA. Amino acid sequence analysis of the Tspans highlights conserved residues which may be critical to tetraspanin structure and function. The Tspans are differentially expressed in human tissues.

CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responsesPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMaecker, H. T., Do, M. S., Levy, S.1998; 95 (5): 2458-2462

Abstract

Mice lacking CD81 (TAPA-1), a widely expressed tetraspanin molecule, have impaired antibody responses to protein antigens. This defect is specific to antigens that preferentially stimulate a T helper 2 response (ovalbumin or keyhole limpet hemocyanin in alum) and is only seen with T cell-dependent antigens. Absence of CD81 on B cells is sufficient to cause the defect. Also, antigen-specific interleukin (IL) 4 production is greatly reduced in the spleen and lymph nodes of CD81-null mice compared with heterozygous littermates. Thus, expression of CD81 on B cells is critical for inducing optimal IL-4 and antibody production during T helper 2 responses. These findings suggest that CD81 may interact with a ligand on T cells to signal IL-4 production. By using a soluble form of CD81 as a probe, a putative ligand for CD81 was identified on a subset of B and T cells. Two possible models for the interaction of CD81 on B cells with a potential ligand on either B or T cells are proposed.

Abstract

CD81 (TAPA-1) is a widely expressed cell-surface protein involved in an astonishing variety of biologic responses. It has been cloned independently several times for different functional effects and is reported to influence adhesion, morphology, activation, proliferation, and differentiation of B, T, and other cells. On B cells CD81 is part of a complex with CD21, CD19, and Leu13. This complex reduces the threshold for B cell activation via the B cell receptor by bridging Ag specific recognition and CD21-mediated complement recognition. Similarly on T cells CD81 associates with CD4 and CD8 and provides a costimulatory signal with CD3. In fetal thymic organ culture, mAb to CD81 block maturation of CD4-CD8- thymocytes, and expression of CD81 on CHO cells endows those cells with the ability to support T cell maturation. However, CD81-deficient mice express normal numbers and subsets of T cells. These mice do exhibit diminished antibody responses to protein antigens. CD81 is also physically and functionally associated with several integrins. Anti-CD81 can activate integrin alpha 4 beta 1 (VLA-4) on B cells, facilitating their adhesion to tonsilar interfollicular stroma. Similarly, anti-CD81 can activate alpha L beta 2 (LFA-1) on human thymocytes. CD81 can also affect cognate B-T cell interactions because anti-CD81 increases IL-4 synthesis by T cells responding to antigen presented by B cells but not by monocytes. The tetraspanin superfamily (or TM4SF) includes CD81, CD9, CD37, CD53, CD63, CD82, CD151, and an increasing number of additional proteins. Like CD81, several tetraspanins are involved in cell adhesion, motility, and metastasis, as well as cell activation and signal transduction.

Abstract

The idiotype (Id) of the Ig expressed on the surface of non-Hodgkin's lymphoma cells is a suitable target for immunotherapy. Indeed, treatment with monoclonal anti-Id antibodies (Abs) can induce long-lasting clinical remissions. However, some of the treated patients relapse with a tumor expressing Ig with point mutations in the idio recognized by the particular monoclonal antibody (MoAb). The alternative approach of active immunization with tumor Id can cure the disease in mice with established tumors and is now being studied in clinical trials. Here, we tested the hypothesis that active immunization with the idiotype would evoke a polyclonal immune response that would cover mutated tumor variants. As a test system, we chose the tumor from a patient who had achieved a complete remission after therapy with anti-Id Ab but subsequently relapsed with a mutated tumor variant no longer binding the treatment Ab. Mice were immunized with proteins and genetic vaccines derived from the original tumor, including (1) Id-keyhole limpet hemocyanin protein, (2) Id single-chain variable fragment (scFv) granulocyte-macrophage colony-stimulating factor (GM-CSF) protein, (3) DNA encoding the Id, and (4) an adenovirus encoding the Id. All immunized mice developed a specific immune response detecting tumor-derived Id proteins from the original tumor and from all tumor variants. We conclude that active immunization with tumor Id can induce a polyclonal immune response and therefore may prevent the escape of mutated tumor variants.

Abstract

DNA vaccination may work through direct transfection of antigen presenting cells (APC), or by secretion of the encoded protein by muscle or skin cells for uptake by APC. If cytokines are attached to the antigen, they may influence APC or responding T cells to drive the response toward a Th1 or Th2 direction, and/or potentiate it in an antigen-specific manner. To test this concept, expression vectors were constructed containing the ovalbumin (OVA) gene either alone, or linked to cytokine genes including GM-CSF, IFN-gamma, IL-2, IL-4, IL-12, or a sequence encoding nine amino acids of IL-1 beta. These constructs expressed OVA-cytokine fusion proteins in vitro which retained cytokine bioactivity. C57BL/6 mice were injected intramuscularly with the DNA constructs. Little if any OVA-specific antibody was produced in response to any of the DNA constructs, except for OVA-IL-4. However, lymphocytes from BALB/c mice vaccinated with OVA-IL-12 and OVA-IL-1 beta constructs produced more IFN-gamma and less IL-4 during in vitro restimulation assays than did other groups. All constructs elicited OVA-specific cytotoxic responses which were maintained or even increased over 16 weeks. The OVA-IL-12 and OVA-IL-1 beta peptide constructs elicited the strongest cytotoxic responses at 2 weeks postinjection. Cytotoxic responses were seen in all animals, even those lacking OVA-specific Ab, and were not related to Ab level. These studies indicate that the humoral, cytokine, and cytotoxic responses to DNA vaccination can be effectively altered by certain cytokine fusion constructs.

Abstract

A legacy of molecular evolution is the formation of gene families encoding proteins that often serve related functions. One such family gaining recent attention is the tetraspanin superfamily, whose membership has grown to nearly 20 known genes since its discovery in 1990. All encode cell-surface proteins that span the membrane four times, forming two extracellular loops. Some of these genes are found in organisms as primitive as schistosomes and nematodes. Alternately known as the transmembrane 4 (TM4) superfamily or the TM4SF, 4TM, or tetraspan family, we propose here that the name tetraspanins be used for the purpose of standardization. What do the tetraspanins do? Awaiting definitive functional studies, we can only put together pieces of a puzzle that has been built by raising antibodies against these proteins and looking at their distribution, associations, and functions. A brief overview indicates that some tetraspanins are found in virtually all tissues (CD81, CD82, CD9, CD63), whereas others are highly restricted, such as CD37 (B cells) or CD53 (lymphoid and myeloid cells). Many of these proteins have a flair for promiscuous associations with other molecules, including lineage-specific proteins, integrins, and other tetraspanins. In terms of function, they are involved in diverse processes such as cell activation and proliferation, adhesion and motility, differentiation, and cancer. We propose that these functions may all relate to their ability to act as "molecular facilitators," grouping specific cell-surface proteins and thus increasing the formation and stability of functional signaling complexes.

Abstract

Optimal treatment of allergic diseases requires that the cytokine profile of allergen-specific T cells be redirected, with the conversion of Th2 profiles into Th1 cytokine profiles. This conversion, however, is difficult, since Th2 effector cells have relatively fixed cytokine profiles. To more effectively redirect the cytokine profiles of T cells, we constructed a cytokine fusion protein that contained the Ag OVA, fused to IL-12. Immunization with the OVA-IL-12 fusion protein induced anti-OVA IgG2a Ab and large quantities of OVA-specific IFN-gamma production. The Ag specificity of this response was dependent upon covalent linkage of Ag and IL-12, since immunization of mice with OVA alone induced little or no IFN-gamma, while immunization with OVA and free rIL-12 enhanced T cell production of IFN-gamma, but the IFN-gamma production was not OVA specific. To examine the effects of OVA-IL-12 in reversing ongoing Th2-dominated immune responses, BALB/c mice previously primed with OVA in alum to induce a Th2-dominated response, were vaccinated with the OVA-IL-12 protein. In such mice, OVA-IL-12 was much more effective than OVA plus free rIL-12 in significantly increasing Ag-specific IFN-gamma production and significantly decreasing Ag-specific IL-4 production. Moreover, OVA-IL-12 increased serum anti-OVA IgG2a and decreased anti-OVA IgE. These studies indicate that OVA-IL-12 can convert immune responses characterized by high IL-4 and high IgE synthesis into Th1-dominated responses in an Ag-specific manner.

Abstract

CD81 is a cell surface molecule expressed on many cell types and associated with the CD19/CD21/Leu13 signal-transducing complex on B cells. A recent report implies that CD81 expression on thymic stromal cells is important in the maturation of thymocytes from CD4-CD8- to CD4+CD8+. However, we have produced CD81-null mice by gene targeting, and find that they undergo normal development of thymocytes and express normal numbers of T cells. B cells are also found in normal numbers in the spleen, blood, and peritoneal cavity of CD81-null mice, but they express a lower level of CD19 compared to heterozygous littermates. Finally, early antibody responses to the protein antigen ovalbumin are weaker in CD81-null mice compared to their heterozygous littermates. This is consistent with the proposed role of the CD19/CD21/CD81-signaling complex in lowering the threshold for B cell responses. These results show that CD81 is not required for maturation of T cells, but is important for optimal expression of CD19 on B cells and optimal stimulation of antibody production.

Abstract

The idiotypic determinants of B cell lymphoma provide a tumor-specific Ag and a target for immunotherapy. We have developed several generations of idiotype vaccines that were tested in an animal model, the 38C13 mouse B cell lymphoma. Initially we showed that effective tumor immunity was elicited by the syngeneic Id when it was conjugated to a carrier protein and mixed with an adjuvant. A subsequent generation of Id vaccines eliminated the need for a carrier protein and for an adjuvant by incorporating cytokines into fusion proteins containing the Id. A third generation of vaccines consisting of naked DNA encoding the Id-granulocyte-macrophage colony-stimulating factor (GM-CSF) fusion proteins was equally effective in inducing tumor immunity. To determine whether Ig variable regions, in the absence of constant regions, could be immunotherapeutic in this model, we tested the use of single-chain Fv (scFv). scFv proteins, produced in bacteria, and naked DNA encoding scFv were used in this study. scFv was tested alone or fused to GM-CSF or an immunoenhancing peptide derived from IL-1beta. Here we demonstrate that scFv-GM-CSF was effective only when injected as a protein, not as a DNA vaccine. In contrast, both scFv-IL-1beta peptide fusion protein and naked DNA encoding it induced tumor immunity that protected mice from tumor challenge.

Abstract

We have previously shown that CD4+ T cells from allergic individuals are predisposed to producing interleukin (IL)-4 in response to allergens. IL-4 production could be modulated by antigen concentration as well as by the type of antigen-presenting cells (APC), with B lymphocytes inducing greater quantities of IL-4 than monocytes. Using this system we examined IL-4 synthesis after culture of CD4+ T cells with B cells, monocytes, or both, as APC in the presence of allergen and a monoclonal antibody against CD81 (TAPA-1), a member of the TM4 superfamily of proteins that regulates activation, proliferation and trafficking of B cells. Addition of anti-CD81 mAb during culture enhanced IL-4 synthesis by 2- to 70-fold over that using an isotype-matched control mAb. Furthermore, anti-CD81 mAb enhanced IL-4 synthesis in CD4+ T cells only when CD4+ T cells were cultured with B cells but not monocytes as APC, indicating that anti-CD81 mAb affected IL-4 synthesis in T cells via interactions with B cells. However, pretreatment of either population separately with anti-CD81 mAb prior to culture had no effect on subsequent IL-4 synthesis, suggesting a requirement for temporal or cooperative interactions between T and B lymphocytes. In addition, anti-CD81 mAb enhanced IL-4 production but reduced CD4+ T cell antigen-specific proliferation, demonstrating that IL-4 production and proliferation by CD4+ T cells were inversely related. Finally, mAb to major histocompatibility complex class II but not to anti-CD19 also enhanced IL-4 synthesis when B lymphocytes were used as APC. In all instances, enhancement of CD4+ IL-4 synthesis correlated with the presence of large cell aggregates in T-B lymphocyte cocultures. These results indicate that the capacity of B cells to induce IL-4 can be significantly enhanced by ligation of particular molecules on their surface and should aid in the design of treatments for diseases in which modulation of the cytokine profile would be beneficial.

Novel unconventional binding site in the variable region of immunoglobulinsPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICARajagopalan, K., Pavlinkova, G., Levy, S., Pokkuluri, P. R., Schiffer, M., Haley, B. E., Kohler, H.1996; 93 (12): 6019-6024

Abstract

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.

THE TAPA-1 MOLECULE IS ASSOCIATED ON THE SURFACE OF B-CELLS WITH HLA-DR MOLECULESJOURNAL OF IMMUNOLOGYSchick, M. R., Levy, S.1993; 151 (8): 4090-4097

Abstract

TAPA-1 is a transmembrane protein that has been shown to be involved in cell growth and cellular adhesion. Our studies were aimed at determining the mechanisms of the biologic phenomena mediated by TAPA-1, which include the identification of proteins that are associated with it on the surface of lymphocytes. We and others have previously shown that Leu-13, a leukocyte Ag, is one such molecule and that in B cells TAPA-1 is associated with the CD19 Ag. Herein we identify an additional molecule, HLA-DR, that is noncovalently associated on the surface of B cells with TAPA-1. This association was first detected by immunoprecipitation by anti-TAPA-1 and by anti-HLA-DR antibodies in the presence of mild detergents. The initial observation was confirmed by 2-dimensional SDS-PAGE and by direct identification of TAPA-1 in anti-HLA-DR immunoprecipitates by Western blot analysis. The association of the two molecules on the surface of a human B cell line was shown by cocapping experiments. In addition, antibodies to both molecules can induce cellular adhesion and an antiproliferative effect. Because the tissue distribution of these two molecules only partially overlaps, with TAPA-1 being expressed on most cell types and MHC class II expressed on a more restricted group of tissue, it is possible that the TAPA-1 molecule provides a basic function that can augment a cell type specific activity. In B cells the association of TAPA-1 with CD19 and HLA-DR may increase cellular interaction and play a supporting role in the transmission of specific signals.

Abstract

We studied the signal induced by the anti-TAPA-1 antibody and compared it to the signal induced by anti-IgM antibodies in a human B cell line, OCl-LY8. We found that exposure of these cells to either antibody resulted in a rapid increase in protein tyrosine phosphorylation which was prevented by inhibitors of tyrosine kinases. Tyrosine phosphorylation was an early event in the cascase leading to the antiproliferative effect of the anti-TAPA-1 antibody. However, 2-ME, a reducing agent that is not an inhibitor of tyrosine kinases, prevented both tyrosine phosphorylation and the antiproliferative effect of the antibody. Cells grown in low concentrations of 2-ME did not exhibit an increase in tyrosine phosphorylation in response to the anti-TAPA-1 antibody and were insensitive to the antiproliferative effect of the antibody. In contrast, the same cells maintained in 2-ME were able to induce tyrosine phosphorylation in response to anti-IgM. The use of 2-ME resulted in an increase in intracellular thiols, mostly glutathione. Moreover, compounds that block glutathione synthesis rendered cells susceptible to the antibody, even in the presence of 2-ME. These experiments demonstrate that tyrosine kinases are involved in propagating the antiproliferative signal initiated by the anti-TAPA-1 antibody and suggest that this signal is dependent upon the level of intracellular thiols.

Abstract

TAPA-1 is a member of a new family of evolutionarily conserved transmembrane proteins which may be involved in regulation of cell growth and/or cell signalling. We have examined the temporal pattern of TAPA-1 RNA expression during mouse development. Using a sensitive reverse transcription/polymerase chain reaction assay, we show that TAPA-1 RNA is present in oocytes, fertilized eggs and cleavage stage embryos.

Abstract

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.

Abstract

Thirty-six randomly selected cases of low grade follicular lymphoma (FL) were analyzed for Ig heavy chain variable region (VH) gene expression. Assignment to one of the six human VH gene families (VH1 to VH6) was made with a polymerase chain reaction-based technique using family-specific leader primers. The frequency of VH family use in FL was found to be similar to that reported for normal peripheral blood lymphocytes and is therefore also roughly proportional to VH family size. To evaluate expression within an individual family, all of the lymphoma VH genes from the middle size VH4 family were sequenced and compared with previously published sequences. Of these eight lymphoma VH sequences, six were most closely related to just two of the 10 known functional VH4 germline genes. Nonrandom usage by FL of the JH3, JH4, and JH5 joining segments was also observed. Nucleotide sequences were also determined for 10 randomly selected lymphoma VH genes from the large VH3 family. With one possible exception, none of these lymphoma VH sequences appear to represent any of the VH3 genes that may be preferentially used in the fetal repertoire.

Abstract

TAPA-1 (the target of an antiproliferative antibody) is a 26-kDa cell surface protein expressed on most human cell lines. TAPA-1 is a member of an evolutionarily related family of cell surface proteins all of which contain four transmembrane domains. A model is proposed for topology of TAPA-1 based on proteolysis studies in the in vitro translated protein embedded into microsomal membranes. This analysis predicts that the amino and the carboxyl termini of the molecule are cytoplasmic and that the two hydrophilic regions of the molecule are extracellular. The antigenic epitope of the human TAPA-1 is contained within a subregion of the second extracellular domain of the protein. This is the only region in the protein that has not been tightly conserved in mammalian evolution.

Abstract

TAPA-1 is a 26-kDa integral membrane protein expressed on many human cell types. Antibodies against TAPA-1 induce homotypic aggregation of cells and can inhibit their growth. The murine homologue of TAPA-1 was cloned from both cDNA and genomic DNA libraries. A very high level of homology was found between human and mouse TAPA-1. The 5' untranslated region of the TAPA-1 gene resembles housekeeping gene promoters with respect to G + C content and the presence of potential Sp1 binding sites. The chromosomal localization of human and murine TAPA-1 genes was determined by Southern blot experiments using DNA from somatic cell hybrids. The genes were found to be part of a conserved syntenic group in mouse chromosome 7 and the short arm of human chromosome 11. The organization of the TAPA-1 gene and the projection of the exon boundaries on the proposed protein structure are presented.

TAPA-1, THE TARGET OF AN ANTIPROLIFERATIVE ANTIBODY, IS ASSOCIATED ON THE CELL-SURFACE WITH THE LEU-13 ANTIGENJOURNAL OF IMMUNOLOGYTakahashi, S., Doss, C., Levy, S., Levy, R.1990; 145 (7): 2207-2213

Abstract

A murine mAb, 5A6 (IgG1), has been isolated by immunization with a human B lymphoma cell line and screening for growth inhibition. The antibody immunoprecipitated a single chain protein of 26 kDa from cell lysates made with Triton X-100 but additional proteins were precipitated when cell lysates were made with the milder detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio)-1-propane sulfate). We have identified one of these coprecipitated molecules as the 16-kDa Leu-13 Ag. 5A6 and anti-Leu-13 showed similar, although not identical, reactivity, growth inhibition and temperature-dependent aggregation effects among hematolymphoid cell lines. The aggregation induced by 5A6 and anti-Leu-13 was not dependent on LFA-1 (lymphocyte function-associated Ag-1). The cell-surface expression of both TAPA-1 (target of an antiproliferative antibody-1) and Leu-13 could be down-modulated by binding to their respective antibodies and they could be reciprocally comodulated. These results suggest that TAPA-1 and Leu-13 form a complex on the cell surface and play a role in growth control through a common pathway.

Abstract

A murine monoclonal antibody was identified by its ability to induce a reversible antiproliferative effect on a human lymphoma cell line. Immunoprecipitation studies revealed that the antibody reacted with a 26-kilodalton cell surface protein (TAPA-1). A diverse group of human cell lines, including hematolymphoid, neuroectodermal, and mesenchymal cells, expressed the TAPA-1 protein. Many of the lymphoid cell lines, in particular those derived from large cell lymphomas, were susceptible to the antiproliferative effects of the antibody. TAPA-1 may therefore play an important role in the regulation of lymphoma cell growth. A cDNA clone coding for TAPA-1 was isolated by using the monoclonal antibody to screen an expression library in COS cells. Analysis of the deduced amino acid sequence indicated that the protein is highly hydrophobic and that it contains four putative transmembrane domains and a potential N-myristoylation site. TAPA-1 showed strong homology with the CD37 leukocyte antigen and with the ME491 melanoma-associated antigen, both of which have been implicated in the regulation of cell growth.

Abstract

mAb secreting hybridomas were produced from mice hyperimmune to the model Ag tobacco mosaic virus protein. Six mAb were selected for their ability to bind synthetic peptides corresponding to amino acid residues 103-112 and 97-107 of tobacco mosaic virus protein. These mAb were analyzed for their fine specificity by measuring binding to synthetic analogs of the decapeptide, and cDNA sequences encoding the mAb V regions were determined. These analyses revealed that a wide range of different V regions are capable of binding with the same decapeptide epitope, and that these antibody sequence differences generally coincided with different binding fine specificities. This diverse antibody response with specificity for the same epitope demonstrates both the breadth of potential of the immune system and the lack of exclusivity in specific protein:protein interactions.

Abstract

The Ig Id of a B cell lymphoma serves as a distinct marker of the malignant clone and thus as a tumor-specific target for antibody therapy. Somatic variation of the Ig genes expressed by B cell tumors can lead to loss of reactivity with anti-Id antibodies and escape of tumors from the therapeutic effects of such antibodies. In our study, we have used anti-Id antibodies to screen for variants within a cell line derived from a patient with a large cell lymphoma of the B cell type. Cells were simultaneously stained on their surface for idiotypic and for isotypic Ig determinants using reagents labeled with different fluorochromes. Tumor cells expressing intact Ig molecules with alteration of their idiotypic determinants were isolated with the fluorescence activated cell sorter. Idiotypic variation was an ongoing process in vitro with Id- variants being generated at a rate of 2.7 x 10(-4)/cell per generation and Ig- cells being produced at a rate of 1.31 x 10(-5)/cell per generation. Subcloned variants expressed subtle differences in reactivity with a panel of three non-cross-blocking anti-Id antibodies. Analysis of Ig gene rearrangements by the Southern blotting technique using a JH probe established that the variants and the original tumor cells were all clonally related. Immunoprecipitation of surface labeled Ig molecules from the variant subclones disclosed major alterations of the lambda-L chains with no gross alterations of the mu-H chains. Related studies have established that the tumor cells undergo rearrangement and expression of new lambda-L chain genes.

Abstract

A murine B cell lymphoma (38C13) was subjected to immunoselection with mAbs directed against the idiotypic determinants of its cell surface Ig. Variants emerged with altered Ig receptors containing identical heavy chains but different light chains. The functional light chain genes in these variants were composed of V kappa segments drawn from the V kappa Ox-1 family, which had replaced the V kappa gene expressed by the parental tumor by rearranging to downstream J kappa segments. Rearrangement at the kappa locus continued to occur spontaneously, giving rise to secondary and tertiary variants at a rate of 1.9 x 10(-4) per cell per generation. Variants were isolated that had ceased production of surface Ig but went on to rearrange again and to become surface Ig+. The Ig- state may be an intermediate step providing a stimulus for continued rearrangement. This process provides an additional mechanism for generating diversity within B cell clones and expands the use of the available repertoire of Ig genes.

Abstract

Mature B cells that express surface immunoglobulin (Ig) are usually committed to their original Ig product. It was shown that such a cell can replace its light chain by rearranging and expressing a new light chain from the other allele. Anti-idiotype antibodies were used to isolate idiotypic variants from a surface IgM+lambda+ human B cell tumor line. The variants expressed a new lambda light chain. Both the original and the new lambda transcripts were present in the variant cells, but only the new one was expressed as a protein on the cell surface. Therefore, although the cell exhibited allelic exclusion and had only one Ig receptor at a time, the commitment to a particular light chain gene was reversible.

Abstract

Idiotype variants of 38C13, a murine B cell lymphoma, have been isolated by immunoselection with antiidiotype mAbs. The V region genes for the kappa light chains and mu heavy chains expressed by these tumor cells were sequenced and compared. There was no evidence for V region somatic point mutation in this tumor. However, while the heavy chain genes were all identical, the light chain genes were all different. The light chain genes of each variant were derived from the V kappa-Ox1 gene family and joined to J kappa 4, whereas the light chain gene of the parental tumor was derived from the V kappa 9 family and joined to J kappa 2. Two of the variants used the identical V kappa gene but differed by the inclusion of a variable number of additional nucleotides in the V/J joint. Thus, the idiotypic heterogeneity of this B cell lymphoma arises as a consequence of alternative light chain rearrangements rather than point mutation. This process repetitively uses members of the same V kappa gene family. Two of the variants use the identical V kappa and J kappa gene segments but differ by the presence of extra nucleotides at the V kappa/J kappa joint.

Abstract

The V region sequence of a non-productive kappa transcript from two myeloma fusion partners has been determined. This transcript has an aberrant VJ recombination site resulting in a translation stop site at position 105. It is variably expressed in hybridomas made from all fusion partners derived from the original MOPC-21 tumor. The amount of this transcript may greatly exceed levels of the productive light chain mRNA.

Abstract

The genes coding for the Ig light chains expressed in two cases of human follicular lymphoma were cloned and sequenced. In each case, multiple independent isolates of the tumor population were compared. Although each tumor represented a single clone of B cells with a unique V/J joint, different cells within each tumor had accumulated multiple point mutations in the V gene during clonal expansion. Most of the mutations observed were silent, but some resulted in amino acid replacements. Identical silent mutations were often observed in independent isolates of each tumor. By combining the current data with VH sequences obtained previously from the same cells, it was apparent that the repetitive silent mutations could not be explained solely by a genealogic tree. Such mutations could represent hot spots whose tendency to mutate may be influenced by neighboring DNA sequences or by the methylation of specific cytosine residues.

Abstract

C3H/HeN mice were immunized with idiotypic immunoglobulin M (IgM) and its molecular subunits from the syngeneic 38C13 lymphoma. Immunization with idiotypic IgM (38C-Id) resulted in idiotype-specific humoral and cellular immunity and protection against a lethal tumor cell challenge. Heavy (H38C) and light (L38C) chains were isolated by electroelution from preparative polyacrylamide gels. Both of these immunogens induced significant resistance to a subsequent tumor challenge. Variable region immunogens, in the form of trpE-fusion proteins, were obtained by cloning heavy and light chain variable region genes into the expression plasmid pATH-11. Of these, only the trpE-VH38C immunogen yielded immune resistance to tumor challenge. Finally, the nucleic acid sequence of 38C-Id light chain was determined and, based on the corresponding amino acid sequence and an analysis of predicted secondary structure, a region of potential antigenicity in complementarity-determining region 3 was chosen for the production of a synthetic peptide. Vaccination with this synthetic peptide resulted in significant suppression of tumor growth. Analysis of the humoral and cellular immunity generated by these vaccines revealed the presence of antibodies reactive with native idiotypic IgM only in 38C-Id, H38C, and trpE-VH38C immune sera, although the latter two were not idiotype-specific. Idiotype-specific lymphocytes, which proliferated in response to native 38C-Id, were observed in all immune animals. With the exception of the fusion protein immunogens, conjugation to an immunogenic carrier protein (keyhole limpet hemocyanin or thyroglobulin) was required for optimal humoral and cellular responses.

RETENTION OF AN IDIOTYPIC DETERMINANT IN A HUMAN B-CELL LYMPHOMA UNDERGOING IMMUNOGLOBULIN VARIABLE-REGION MUTATIONPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAKon, S., Levy, S., Levy, R.1987; 84 (14): 5053-5057

Abstract

Tumor cells from a patient with B-cell lymphoma were fused with a mouse myeloma cell line. A set of heterohybridomas was thus derived, each of which represented a separate clonal derivative from the tumor cell population. The immunoglobulins secreted by these cell lines reacted variably with a panel of anti-idiotypic antibodies, indicating that the tumor was heterogeneous; however, one antibody, 4D6, reacted strongly with the product of all the heterohybridomas. cDNA for the immunoglobulin heavy chain variable-region genes expressed in these heterohybridomas was cloned and sequenced. Comparison of these sequences indicated that the cells expressing them were clonally related but that they had undergone considerable mutation. Despite mutation, the cells in this tumor population continued to express a functional immunoglobulin molecule and to retain, over a span of 3 years, the idiotypic determinant defined by the 4D6 monoclonal antibody. Thus a selective force existed within the host to retain tumor cells bearing an immunoglobulin molecule with a particular idiotypic structure.

Abstract

A rapid procedure is described for cloning immunoglobulin V region genes from cells that express them. cDNA is synthesized from mRNA template using primers homologous to the immunoglobulin constant-region genes. Blunt-ended, double-stranded cDNA is obtained by sequential addition of enzymes to a single tube. The cDNA is inserted directly into the M13 vector, which is screened by plaque lifting for the presence of specific inserts. Screening probes can be generated from 32P-labeled single-stranded cDNAs generated from primers different from those used for cloning, or alternatively, from previously cloned V or C gene segments. The ease of cloning a cDNA V region is directly related to the abundance of Ig-specific mRNA within the cell of interest. This method minimizes the number of steps and the time needed to obtain accurate and complete sequences of any expressed Ig V region gene.

Abstract

Using isolated idiotype (Id) protein we generated panels of antibodies in two patients with follicular lymphoma, one of whom had never received prior chemo-or radiotherapy. Flow cytometry and frozen section tissue staining of tumor with these monoclonal antibodies (mAb) revealed multiple subpopulations within each tumor. Individual mAb stained between 7% and 83% of surface Ig+ cells in the tumor samples. These subpopulations were overlapping and no single antibody recognized all the tumor cells. However, combinations of antibodies seemed to capture total tumor in both cases. In some instances, the percentage of tumor stained by a single mAb varied over time, and differed between lymph nodes sampled at the same time. Because a single species of Id protein was used to generate mAb in each case, it appears that the antibodies were directed against idiotopes variably shared by different populations within each tumor, and this was confirmed by crossblocking studies. Tumor cells from one patient were fused to a nonsecreting heteromyeloma line K6H6/B5, and most of the resulting hybrids secreted Id protein. Four mAb were used to screen the Id proteins secreted by these hybrids, and 11 different variants (16 maximal) were found. Southern blot analysis of rearranged Ig genes was done in two hybrids and biopsy material. Identically rearranged light-chain genes were seen but it appeared as though extensive somatic variation had occurred in heavy chain genes. These studies indicate that: striking Id variation can exist at diagnosis in untreated patients, the percentage of tumor represented by an individual variant may change with time and may differ between tumor sampled from different anatomical locations, and somatic variation appears to be responsible for the observed heterogeneity. Although this degree of variation makes anti-Id antibody therapy more difficult, appropriate combinations of mAb should be more efficacious than single antibodies in such cases.

Abstract

Following treatment of a human B cell lymphoma with an anti-idiotype antibody, a subpopulation of tumor cells remained that had lost the tumor-specific heavy chain idiotypic determinant. Nucleotide sequence analyses of eight independent heavy chain variable region isolates showed extensive point mutations, so that no two sequences were identical. Comparison of pretreatment and posttreatment sequences implicated an amino acid in CDR2 as being involved in the idiotypic determinant. Apparently the malignant B cells escaped the therapeutic effects of the anti-idiotype antibody through an ongoing process of somatic mutation in their immunoglobulin genes. Non-random clustering of amino acid replacements in CDR2 suggested that growth of the tumor may have been influenced by endogenous selective forces interacting with the tumor cell-surface immunoglobulin.

Abstract

The nucleic acid sequence of the heavy chain variable region (VH) expressed by 38C13, a B cell tumor of C3H origin, was determined by a combination of direct (messenger RNA) mRNA sequencing by primer extension and complementary DNA (cDNA) isolation and sequencing in M13. The VH amino acid sequence was deduced, and hypervariable regions were identified. From an analysis of predicted secondary structure, regions of predicted antigenicity were chosen, and a series of synthetic peptides corresponding to CDR2 and CDR3 (complementarity-determining region) were produced. These peptides were coupled to protein carriers and used to immunize syngeneic C3H mice. All peptides gave rise to a vigorous antibody response. However, only the CDR3 peptides induced antibodies that crossreacted with the isolated H chain protein. Only one CDR3 peptide induced antibody-producing clones, isolated as hybridomas, that reacted with the intact IgM protein. However, the appearance of these clones was a low-frequency event. All antibodies reacting with the H chain or the intact IgM protein were idiotypically specific for 38C13. These monoclonal antiidiotype (anti-Id) antibodies, raised against CDR3 peptides, gave strong reactions in enzyme-linked immunosorbent assays and immunoblots, but they were of low affinity compared to syngeneic anti-Id raised against the intact IgM protein. Moreover, while the intact IgM was capable of inducing tumor immunity, the CDR peptides were not able to do so.

Abstract

Sensitive enzyme-linked immunosorbent assays (ELISA) for the detection of human T cell antigens in soluble form have been developed. The assays use mouse monoclonal antibodies and specific anti-Leu sera prepared in rabbits by immunizing with Leu antigens absorbed to monoclonal antibody affinity columns. With these assays, Leu-1, -2, and -3 antigen signals from extracts of as few as 5 X 10(3) cells could be detected. When culture supernatants from various cell lines were tested, Leu-2 antigen, but not Leu-1 or Leu-3, was found to be present. Leu-2 antigen was present only in supernatants from T cell lines that expressed Leu-2 on their cell surface. Leu-2 antigen accumulated progressively in the supernatant of low density culture and its presence did not depend on cell proliferation or on fetal calf serum in the culture medium. The Leu-2 antigen in the supernatant was found to have only one Leu-2a determinant, whereas Leu-2 antigen from cell extracts had at least two determinants. The Leu-2 molecule was effectively purified from supernatant with an anti-Leu-2a affinity column. The purified Leu-2 antigen from supernatant of HPB-ALL cells was a single polypeptide chain of 27,000 mol wt, whereas Leu-2 antigen present on HPB-ALL cell surface was composed of two or more identical polypeptide chains of 33,000 mol wt linked by disulfide bonds. Normal human sera and sera from leukemia patients were also examined for the presence of the Leu-2 antigen. Normal human sera contained low levels of Leu-2 antigen but sera from Leu-2-positive leukemia patients had high levels. These results indicate that Leu-2 antigen is released from human T cells under physiological conditions.

THE NUCLEOTIDE AND AMINO-ACID CODING SEQUENCE OF A GENE FOR H-1 HISTONE THAT INTERACTS WITH EUCHROMATIN - THE EARLY EMBRYONIC H-1 GENE OF THE SEA-URCHIN STRONGYLOCENTROTUS-PURPURATUSJOURNAL OF BIOLOGICAL CHEMISTRYLevy, S., Sures, I., Kedes, L.1982; 257 (16): 9438-9443

Abstract

We have determined the nucleotide sequence of the gene that codes for an H1 histone associated with euchromatin in the sea urchin Strongylocentrotus purpuratus. The gene codes for a protein of 205 amino acids. The nucleotide sequence of this gene is homologous to the sequence of H1 mRNA that is expressed during early embryonic development. We have compared the nucleotide and protein coding sequences of this H1 gene to those of the early H1 histone gene of the sea urchin Psammechinus miliaris. There is considerable drift by nucleotide substitution between the two genes randomly distributed across the mRNA coding region. Despite this divergence of nucleotide sequence, there are local constraints on amino acid substitutions throughout the molecule and especially in its central region. We have also compared the amino acid sequence in this central hydrophobic region of the euchromatic S. purpuratus H1 histone to the same region in H1 and H5 histones associated with heterochromatin. We show that certain amino acids are conserved and aligned in frameworks in all the sequenced H1 proteins.

Abstract

We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefore cannot originate from the spacers present in the histone genes. We conclude that there is no detectable transcription of the spacer regions on either side of the H2B histone gene. The detection limit for RNA complementary to the 5' spacer sequence corresponds to a maximum of about three RNA molecules per cell, an amount shown to be far less than the projected steady-state pool size of a putative polycistronic transcript, if such a precursor were to be the obligatory transcript of the histone genes. (This conclusion was derived by using the known rates of production of H2B mRNA throughout early development [R. E. Maxson and F. H. Wilt, Dev. Biol., in press].) The physiologically relevant transcript of the histone genes in early development is therefore monocistronic and probably identical to the messenger RNA itself.

LEADER SEQUENCES OF STRONGYLOCENTROTUS-PURPURATUS HISTONE MESSENGER-RNAS START AT A UNIQUE HEPTANUCLEOTIDE COMMON TO ALL 5 HISTONE GENESPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESSures, I., Levy, S., Kedes, L. H.1980; 77 (3): 1265-1269

Abstract

We have determined the sequence of the untranslated leader nucleotides of all five histone mRNAs from Strongylocentrotus purpuratus by the dideoxy chain termination method. Total polysomal RNA from sea urchin embryos was used as a substrate for cDNA synthesis primed by specific DNA restriction fragments. Each of the primers was derived from the 5'-terminal part of the coding region for a different histone protein. The five histone mRNA leader sequences are different in length and primary structure. The 5' termini of all five histone mRNAs coincide with the unique heptanucleotide Py-Py-A-T-T-C-Pu in genomic DNA. This sequence, which defines the start of the individual histone mRNAs, is preceded by the A+T-rich octanucleotide identified in front of all eukaryotic structural genes where sequences have been determined to date.

Abstract

We describe a rapid and simple method for the purification of biologically active messenger RNAs. The method allows the isolation in a few hours of specific mRNAs from either whole cell or polysomal RNA even if the RNA represents less than 1% of the starting molecules. We used, as a model, cloned sea urchin (Strongylocentrotus purpuratus) histone gene fragments linked to cellulose by the method of B. E. Noyes & G. R. Stark (1975) Cell 5, 301--310) as hybridization probes to isolate specific histone mRNAs from whole cell and polysomal RNA extracts. RNAs isolated in this manner maintain their biological activity, serving as templates for histone proteins in a wheat-germ, cell-free protein translation system. In addition, radiolabeled histone-specific RNA purified from cleavage stage sea urchin embryos, pulse labeled for short periods of time and analyzed on denaturing polyacrylamide gels, was the same size as mature histone mRNA's.

Abstract

RNA transcribed in isolated sea urchin nuclei and assayed by hybridization to histone genes cloned in E. coli contains sequences homologous to each of the five histone genes. Histone RNA is synthesized exclusively from the same DNA strand which is the template in vivo. Synthesis of the histone gene transcripts is sensitive to alpha-amanitin concentrations which inhibit RNA polymerase II activity. The fraction of histone RNA synthesized in vitro is comparable at two developmental stages to the fraction synthesized in vivo. The nuclear histone transcripts contain sequences homologous to spacer DNA regions present between the coding regions of the 6500 base pair (bp) histone gene repeat unit. The transcription of spacer sequences was demonstrated by hybridization of the nuclear transcripts to subcloned spacer DNA. Although the bulk of the RNA transcripts are greater than 2000 bases long, the histone-specific transcripts are of discrete sizes ranging from 100 bases to about 1100 bases long. Each histone gene hybridizes with at least one of the larger transcripts and with a different subset of smaller RNAs. We do not detect any giant polycistronic transcript spanning the entire histone repeat unit.

Abstract

Newly synthesized polysomal messenger RNAs from cleavage stage embryos of the sea urchin Arbacia punctulata and Lytechinus pictus that contain putative histone mRNAs have been fractionated on 6% polyacrylamide slab gels. At least 8 RNA species with unique electrophoretic mobilities have been recognized. The complex of RNAs has been eluted from the gels in three groups, A, B, and C, in increasing order of mobility. The template activity of the three fractions and the unfractionated starting material was examined in the mouse Krebs II ascites tumor cell-free protein synthesizing system. The unfractionated messenger complex programs the synthesis of proteins that co-electrophorese exclusively with sea urchin histones in both sodium dodecyl sulfate and acid urea gel systems. The products of in vitro protein systhesis stimulated by the individual polyacrylamide gel RNA fractions were similarly examined. Each stimulated protein synthesis and was enriched for specific histone templates. We conclude that RNA fraction A is template for histone f1, C is template for histone f2a1, and B serves as template for f2b, f2a2, and f3 histones. A minor degree of contamination of the A and B RNA fractions was obvious from the production of other histones by each template. The co-electrophoresis of specific template activity with specific radiolabeled RNAs supports the concept that most or all of the labeled RNAs are indeed themselves the histone mRNAs.